The primary function of the immune system relates to the protection of the body from disease. The immune system protects against not only those diseases which result from an attack by bacteria, virus, and other pathogens, but also cancer, as well as disease states which result from immune imbalance; opportunistic infections, or autoimmune disorders.
In a number of diseases or other pathological conditions, the immune system response of an animal or human is depressed. As a result, the subject becomes more susceptible to malignancies, or pathological infections against which a normal immune system would have protected the subject. Some conditions which depress the immune system include acquired immune deficiency syndrome (AIDS), chronic infection, chemotherapy, trauma of surgery, and aging.
Modulation of the immune system through pharmaceutically induced stimulation or suppression offers an important approach to the control of disease. Compounds which nonspecifically stimulate the immune system are of potentially significant medicinal importance and have been the object of lengthy research efforts. Often, the research results show that immunomodulating compounds are either weak immunostimulants, and therefore not very effective, or potent immunostimulants and, therefore, effective but toxic by virtue of this potent immunostimulating activity.
Among the many classes of compounds which non-specifically stimulate the immune system are a number of naturally occurring peptides or fragments thereof. One such fragment is Imreg, a tripeptide of the general sequence Tyr-Gly-Gly disclosed in U.S. Pat. No. 4,699,898 issued Oct. 13, 1987. This patent does not disclose the sequence of the peptides of the present invention. Imreg can be isolated from human leukocytes. It is a thymomimetic but possesses weak potency. (A. Arthur Gottlieb, Int.J.Immunopharm.Vol.13, Suppl.1, p.29-32 (1991)).
Another fragment of a naturally occurring peptide is the tetrapeptide Tuftsin and its close analogue Rigin. These peptides have the respective sequences Thr-Lys-Pro-Arg and Gly-Gln-Pro-Arg. Analogues of tuftsin can be found, for example, in Biondi et al., "Synthesis of glycosylated tuftsins and tuftsin-containing IgG fragment undecapeptide", Int.J.Peptide Protein Res. 37, 1991, p.112-121, and Verdini et al., "Immunostimulation by a partially modified retro-inverso-Tuftsin analogue containing Thr.sup.1 .psi.[NHCO](R,S)Lys.sup.2 modification", J.Med.Chem., 1991 34, p.3372-3379. These publications do not disclose peptides with the sequence of the present invention. Tuftsin and rigin are both fragments of IgG and are found within the heavy chain of leukokinin. Tuftsin is a well-known macrophage activator and is known to stimulate NK cell activity. However, it is unstable in plasma which may strongly reduce its stimulating effects on the immune system.
Another fragment of a naturally occurring peptide known to have immunomodulating effects is thymopentin. (Audhya et al., "Contrasting biological activities of thymopoietin and splenin, two closely related polypeptide products of thymus and spleen", Proc.Natl.Acad.Sci.USA, 81,p.2847-2849, 1984; U.S. Pat. No. 5,218,089 issued Jun. 8, 1993; U.S. Pat. No. 5,091,510 issued Feb. 25, 1992). This pentapeptide has the sequence Arg-Lys-Asp-Val-Tyr. Thymopoietin is a 49 amino acid polypeptide thymus hormone. The pentapeptide is the smallest fragment of thymopoietinq which retains the immunological activity of the parent hormone. Thymopentin is a thymomimetic of weak potency. It induces phenotypic differentiation of T cells and increases Interleukin-2 production. Splenin, an analogue of thymopentin, affects both B and T cells. Splenin has the sequence Arg-Lys-Glu-Val-Tyr in bovine and Arg-Lys-Ala-Val-Tyr in humans. Another similar peptide is thymotrinan which has the sequence Arg-Lys-Asp. While these peptides also produce immunomodulating effects, they do not contain or disclose the sequence of the peptides of the present invention.
Another naturally occurring compound is Eisenin extracted from a brown marine algae. This tripeptide has the sequence pyroGlu-Gln-Ala. It was reported in Kojima et al., "Eisenin (L-pyroGlu-L-Gln-L-Ala), a new biological response modifier", Journal of Immunotherapy, 13, p.36-42, 1993 that Eisenin showed immunological activity augmenting natural cytotoxicity of peripheral blood lymphocytes (PBLs) in humans. The natural cytotoxicity augmented by Eisenin seems to be due mainly to NK cells.
One peptide named "LANT 6" has the sequence Lys-Asn-Pro-Tyr-Ile-Leu. LANT6 has been found in ganglion cells and is believed to play a role in neurotransmission between retina ganglion cells and their central target areas. LANT 6 possesses some sequence homology with neurotensin. Neurotensin analogues have been described as cerebral medicaments, antipsychotic agents, analgesics, and anphetamine antagonists. However, LANT 6 has not been reported to have immunomodulatory activity.
Furthermore, it is known in the peptide art, as noted in U.S. Pat. No. 4,426,324 "Immunopotentiating Peptides" issued Jan. 17, 1984, that deletion of even one amino acid from the sequence of a biologically active peptide can result in the loss of biological activity. Therefore, the inclusion of the sequence of the present invention in the sequence of LANT 6 for use as a neuropeptide does not indicate that a shorter sequence within LANT6 would be active as an immunomodulator.
It is well known that vaccines are important in the prophylaxis of disease. Vaccines operate by exposing a host animal to foreign material designed to activate the immune system to confer upon the host immunity against the material without exposing the host to the risk of disease. At the present time, about 20 vaccines have been developed for commercial use. Some of these vaccines are made by detoxification of the toxin produced by the microorganism, or a portion of that organism, or by isolation of a specific non-toxic portion of the organism. A well known example of the latter is the isolation of capsular polysaccharides from meningococcal and pneumococcal bacteria as the basis for a vaccine for bacterial meningitis and pneumonia. However, polysaccharide vaccines are poor immunogens which do not give rise to adequate amounts of protective antibodies in individuals with poorly developed or impaired immune systems. The latter includes young children, the elderly, or those with autoimmune diseases. Furthermore, the immune response which does occur is T-independent or non-memory which means the individual will not display an increased antibody response, with seroconversion, upon being given a booster injection. T-cell dependence is necessary for the induction of IgG antibodies and memory cells. Thus, upon seroconversion, both IgM and IgG antibodies are formed upon repeated injections of a vaccine. Furthermore, the magnitude of the antibody response increases with each injection of vaccine, when the response is T-dependent. The immunology of polysaccharide vaccines has been reviewed by Kennings et al, "The Polysaccharides" (Editor; GO Spinal), Volume 1, 291-329 (1982).
As regards adjuvants for commercial use, only aluminum and calcium salts are presently employed as adjuvants. However, aluminum and calcium salts are not potent adjuvants. Calcium salts have found only limited use. While aluminum salts have found more widespread use with other vaccines, little success has been reported with polysaccharide protein conjugate vaccines. In fact, it has been reported that aluminum hydroxide inhibits the antibody response to an H. Influenzae b polysaccharide-tetanus toxoid conjugate vaccine; J. B. Robbins, et al also observe the same suppression of the antibody response with aluminum hydroxide and a S Typhi polysaccharide--cholera toxin conjugate vaccine; J. Experimental Medicine, 166, 1510-1524 (1897). Furthermore, aluminum salts may provoke transient or chronic local granulomas at the site of injection; L. H. Collier in lancet, 1354-1367 (1987) states that the incidence and severity of reactions to tetanus toxoid vaccine depends upon the presence of aluminum adjuvants. The preparation of aluminum adjuvants is not always reproducible. Moreover, aluminum can alone stimulate the production of IgE antibodies which are responsible for mediating immediate hypersensitive reactions. This has been described by T. Matuhasi et al, J. Infectious Disease, 146, 192 (1982).
Attention has focused in recent years on the use of organic compounds as immunoadjuvants. Only a few organic compounds function in a manner similar to commercially acceptable aluminum salts; i.e. as a slow release vehicle or antigen (vaccine) depot whereby antigen is released over a relatively long period of time at the site of injection.
Examples of such organic compounds are organic surfactants and emulsifiers, such as Pluronics and Tetronics which are non-ionic block copolymers of polyoxyethylene and polyoxypropylene produced by BASF Corporation. Such a slow-release mechanism of adjuvanticity has long been accepted for human use since it reduces the possibility of overstimulating the immune system. Overstimulation of the immune system can lead to an autoimmune response such as would occur with the use of a potent immunostimulant, for example Freund's adjuvant. Thus, the slow release mechanism is the preferred mechanism.
While the majority of organic adjuvants have been shown to be potent immunostimulants, such highly active adjuvants tend to be toxic and therefore unacceptable for human use. Examples of known organic adjuvants which are potent immunostimulants are Freund's complete adjuvant and muramyl dipeptide. Both of these compounds are restricted to use in animal research because of toxicity considerations. Many of the organic adjuvants which mimic aluminum salts are more toxic than aluminum salts. For example, long chain alkyl amines described by D. Gall in Immunology, 11, 369-386 (1966) are reported to be toxic compounds which are generally disruptive to cell membrane structure.